Cocaine Augments Dopamine-Mediated Inhibition of Neuronal Activity in the Dorsal Bed Nucleus of the Stria Terminalis

Abstract

The dorsal region of the bed nucleus of the stria terminalis (dBNST) receives substantial dopaminergic input which overlaps with norepinephrine input implicated in stress responses. Using ex vivo fast scan cyclic voltammetry in male C57BL6 mouse brain slices, we demonstrate that electrically stimulated dBNST catecholamine signals are of substantially lower magnitude and have slower uptake rates compared with caudate signals. Dopamine terminal autoreceptor activation inhibited roughly half of the catecholamine transient, and noradrenergic autoreceptor activation produced an ∼30% inhibition. Dopamine transporter blockade with either cocaine or GBR12909 significantly augmented catecholamine signal duration. We optogenetically targeted dopamine terminals in the dBNST of transgenic (TH:Cre) mice of either sex and, using ex vivo whole-cell electrophysiology, we demonstrate that optically stimulated dopamine release induces slow outward membrane currents and an associated hyperpolarization response in a subset of dBNST neurons. These cellular responses had a similar temporal profile to dopamine release, were significantly reduced by the D2/D3 receptor antagonist raclopride, and were potentiated by cocaine. Using in vivo fiber photometry in male C57BL/6 mice during training sessions for cocaine conditioned place preference, we show that acute cocaine administration results in a significant inhibition of calcium transient activity in dBNST neurons compared with saline administration. These data provide evidence for a mechanism of dopamine-mediated cellular inhibition in the dBNST and demonstrate that cocaine augments this inhibition while also decreasing net activity in the dBNST in a drug reinforcement paradigm.SIGNIFICANCE STATEMENT The dorsal bed nucleus of the stria terminalis (dBNST) is a region highly implicated in mediating stress responses; however, the dBNST also receives dopaminergic inputs from classically defined drug reward pathways. Here we used various techniques to demonstrate that dopamine signaling within the dBNST region has inhibitory effects on population activity. We show that cocaine, an abused psychostimulant, augments both catecholamine release and dopamine-mediated cellular inhibition in this region. We also demonstrate that cocaine administration reduces population activity in the dBNST, in vivo Together, these data support a mechanism of dopamine-mediated inhibition within the dBNST, providing a means by which drug-induced elevations in dopamine signaling may inhibit dBNST activity to promote drug reward.

Keywords: BNST; cocaine; dopamine; electrophysiology; fiber photometry; voltammetry.

Figure 1.

Electrically stimulated catecholamine release in the dBNST ex vivo. A. Schematic representation of coronal sections containing the BNST (green). Electrically stimulated catecholamine release was measured in the dBNST (1, purple) as well as in the caudate (2, gray) for planned comparison. B, Representative voltammetry trace of catecholamine release in dBNST showing current versus time (top), corresponding color plot (bottom), and voltammogram (inset). Scale bar: current (nA) converted to catecholamine (250 nM) versus time (2 sec). C, Examples of stimulated catecholamine release measured in dBNST (top, 10 pulse stimulation train) and caudate (bottom, 1 pulse stimulation). Grouped data showing catecholamine release (D) and uptake rates (E) in the BNST are lower compared with the neighboring caudate. Error bars indicate +/- SEM. *p < 0.05. F, Correlation analysis, fitted with linear regression line (blue), showing positive relationship between catecholamine release and uptake rate in the dBNST. G, Correlation analysis, fitted with linear regression line (blue), showing positive relationship between catecholamine release and uptake rate in the caudate.

Figure 2.

Dorsal BNST catecholamine autoreceptor pharmacology. A, Grouped data timeline showing the cumulative effects of the D2 autoreceptor agonist quinpirole (Quin, arrow) and the α2 adrenergic autoreceptor agonist UK 14304 (UK, arrow), applied in series. Each autoreceptor agonist provided distinct inhibition of the stimulated release magnitude over the course of the experiment. B, Representative traces of the dBNST signal at baseline (BL, black) and following bath application of Quin (blue) and subsequent application of UK (Quin + UK, red). Each autoreceptor agonist reduced the amplitude of catecholamine release. C, Grouped data showing the amount of inhibition resulting from Quin (blue) was greater than inhibition from UK (red). D, Estimated contribution of dopamine (DA, blue) and norepinephrine (NE, red) to the total catecholamine signal in the dBNST, based on magnitude of autoreceptor agonist effects. The majority of the catecholamine signal in the dBNST is DA. E–H, Reversing the order of autoreceptor agonist application results in increased effects of UK on stimulated catecholamine signals. E, Timeline showing the cumulative effects of UK and Quin application on stimulated catecholamine signals. F, Representative traces of stimulated catecholamine signals at BL (black), following application of UK (red), and subsequent application of Quin (UK + quin, blue). G, Grouped data showing the respective signal inhibition produced by UK and Quin. H, The estimated contribution of DA is greater than NE in the dBNST. I–L, Autoreceptor agonist pharmacology in the ventral BNST. I, Timeline of Quin and UK application on stimulated catecholamine signals in the ventral BNST. J, Representative traces of ventral BNST catecholamine signals at BL (black), following application of Quin (blue) and subsequent application of UK (Quin + UK, red). K, Grouped data showing the respective signal inhibition produced by Quin and UK in the ventral BNST. L, Estimated contribution of DA and NE to catecholamine signals in the ventral BNST; catecholamine signals in the ventral BNST are primarily noradrenergic. Grouped data presented as mean, error bars indicate +/- SEM; individual data points overlain on bar graphs. *p < 0.05.

Figure 3.

Cocaine effects on dBNST catecholamine signals, ex vivo. A, Representative traces demonstrating the effects of bath-applied cocaine on stimulated catecholamine release in the dBNST. Baseline (BL, black) signals showed increased release and duration in the presence of cocaine (1 μm, maroon; 10 μm, blue). B, Grouped data show that cocaine increased the catecholamine signal half-life in a dose-dependent manner. *p < 0.05 versus baseline. Data fit with a nonlinear regression line (black). C, Representative traces demonstrate that the DAT-specific uptake inhibitor, GBR 12909 (1 μm, gray), increased catecholamine release and duration in the dBNST, similar to cocaine. D, Grouped data show that GBR 12909 (1 μm, gray) nearly doubled the catecholamine signal half-life in the dBNST. *p < 0.05. Grouped data presented as mean with individual points overlain; error bars indicate +/- SEM.

Figure 4.

Dopamine receptor mRNA expression across dBNST neurons. A, Dopamine mRNA transcript binding in the dBNST. D1 receptor (red) and D2 receptor (green) mRNA transcript expression is robust in the caudate region around the dBNST, whereas, within the dBNST, receptor transcript expression is comparatively less abundant. B, Higher magnification (40×, gray box in A) of dBNST dopamine receptor transcript expression shows that D1 and D2 receptor mRNA is detected in numerous dBNST neurons, as denoted by transcripts overlapping with DAPI staining (blue). C, High magnification (63×) shows that D1 and D2 receptor mRNA expression is largely segregated across separate populations of dBNST neurons. D, Quantification of D1 and D2 receptor mRNA expression across neurons in the lateral half of the dBNST (n = 9 unilateral dBNST across 5 animals). D1 (red) and D2 (green) receptor transcripts were detected in ∼30% of neurons, and were largely segregated to distinct populations (2.9% of cells with overlapping expression; yellow). There was a similar proportion of expression of D1 (17.6%) and D2 (13.7%) transcripts across cells (p = 0.13), with ∼65% of dBNST neurons showing no expression of dopamine receptor transcripts (unlabeled, blue).

Figure 5.

Optically stimulated dopamine signals in the dBNST. A, Histologic assessment of viral injections targeting dopamine neurons in the VTA. A-C, Red represents TH expression. Green represents ChR2 expression. B, Viral injections targeting dopamine neurons in the CLi and the vPAG. C, Viral injections in dopamine A10 and A10dc cell groups (A and B) results in robust and targeted ChR2 expression in dopamine terminal fields in the dBNST. D, Representative cyclic voltammetry trace of a dopamine signal elicited with a 20 pulse, 20 Hz optical stimulation train (blue). Top, Current increased in response to optical stimulation and lasted ∼10 s. Inset, Current peaks at 0.6 V and −0.2 V reflect the oxidation and reduction voltages for dopamine. Bottom, Color plot represents current (z axis) as a measure of voltage (y axis) and time (x axis). Scale bar: y = 100 nm dopamine, x = 1 s. E, Optically stimulated dopamine release magnitude was consistent across successive 20 pulse stimulations, applied every 10 min (20p, black line). Stereotyped reductions in release magnitude occurred when stimulation trains were reduced to 10 pulses (10p), 5 pulses (5p), or a single pulse (1p); release magnitude was consistent across repeated stimulations, applied every 5 min, for 10p and 5p stimulation parameters (black line). Application (Drug, arrow) of the D2 autoreceptor agonist quinpirole (quin, red) inhibited release across all stimulation parameters, whereas application of the D2 autoreceptor antagonist raclopride (Rac, purple) increased release with longer duration stimulation trains (20p). Data are grouped average, error bars indicate +/- SEM.

Figure 6.

Dopamine-mediated hyperpolarization responses in dBNST neurons. A, Representative trace of dopamine release in response to optical stimulation (10 pulses, 20 Hz; blue). B, Representative averaged trace of slow outward current (pA) in response to optically stimulated dopamine release (n = 7 cells). C, Representative averaged trace of hyperpolarizing voltage (mV) response to optical stimulation at resting membrane potential, normalized for grouped average (n = 7 cells). D, Grouped data showing the absolute peak amplitude of current (pA) and voltage (mV) responses across cells. E, Grouped data showing the signal half-life for current (pA; B) and voltage (mV; C) responses across cells. F, The D2/D3 receptor antagonist raclopride (2 μm) significantly reduced the peak hyperpolarizing voltage (mV) amplitude in response to optical stimulation. *p < 0.05. G, Input resistance as a percentage of baseline resistance is significantly decreased following optical stimulation in cells with a slow current response (SC⁺) compared with cells with no slow current response (SC^–). *p < 0.05. Bar graphs represent grouped mean with individual points overlain; error bars indicate +/- SEM.

Figure 7.

Glutamate corelease from optically stimulated dopamine terminals in the dBNST. A, Representative trace of optically stimulated dopamine-induced cellular response highlighting the glutamate corelease upon stimulation (red box). Inset, Example trace of a cell with strong glutamate corelease with clear EPSCs detected at each pulse of the stimulation train (blue). B, Grouped data showing the magnitude of EPSCs detected across the 10-pulse stimulation train for all cells that showed detectable glutamate corelease. The magnitude of first pulse EPSCs was greater than the EPSC magnitude for each subsequent pulse in the stimulation train. *p < 0.001. C, There was no correlation between the amplitude of the first pulse EPSC (when present) and the magnitude of slow current responses, suggesting that these events are not interdependent.

Figure 8.

Cocaine augments dopamine-mediated inhibitory responses in dBNST neurons. A, Representative trace showing cocaine effects on optically stimulated (blue; 10 pulses at 20 Hz) slow current responses; data reduced to 100 Hz sampling frequency to highlight differences in slow current. Cocaine (red) extended the duration of slow currents compared with baseline (black, n = 2 cells averaged). B, Representative trace showing that cocaine (red) extends the duration of optically stimulated hyperpolarization responses compared with baseline (black, n = 6 cells averaged). C, Cocaine did not change the peak amplitude of slow current responses (pA). D, Grouped data showing that cocaine significantly increased the slow current (pA) half-life compared with baseline (n = 5 cells). *p < 0.05. E, Cocaine did not alter the peak amplitude of hyperpolarization responses (mV). F, Grouped data showing that cocaine significantly increased the half-life of hyperpolarization responses (mV) compared with baseline (n = 7 cells). *p < 0.01. G, Example trace of a cell firing endogenous action potentials in response to cocaine; the optically stimulated dopamine-mediated hyperpolarization response paused endogenous spiking. Bar graphs represent grouped mean with individual point overlain; error bars indicate +/- SEM.

Figure 9.

Cocaine administration reduced calcium transient activity in dBNST neurons during acquisition of cocaine CPP. A, Cocaine CPP experimental design. Cocaine (blue) or saline (white) was administered intraperitoneally and paired with a distinct side of the CPP chamber across four training sessions for each treatment (top). Before training, animals had surgeries to express GCaMP in dBNST neurons (bottom left) and had a chronically implanted fiber optic ferrule positioned over the dBNST. In vivo fiber photometry (bottom right) was used to record endogenous GCaMP activity on the first and fourth training sessions for cocaine and saline (top left, green). B, Post hoc analysis of optical fiber placements in the dBNST for fiber photometry recordings of GCaMP activity. C, Grouped data showing cocaine administration significantly increased locomotor activity during the first and fourth training sessions compared with the corresponding training sessions with saline administration. D, Cocaine significantly decreased the frequency of GCaMP activity in the dBNST during the first and fourth training session compared with saline administration. E, Cocaine place conditioning increases time spent on the cocaine-paired side during the post-conditioning test (POST) compared with the pre-conditioning (PRE) baseline. Bar graphs represent grouped mean with individual points overlain; error bars indicate +/- SEM. **p < 0.0001.